Not applicable.
The present invention is in the field of biotechnology and specifically relates to the study of the physiological and physiochemical processes which govern and underlie the formation, growth and resorption of human and animal bone. In particular the invention provides novel means for the study of responses of the mammalian musculoskeletal system to stress and potentially may lead to the discovery of novel substances produced by bone during these responses. The instant system may lead to a better understanding of diseases such as osteoporosis and the perfusion chamber means provides means for the study of the effects of drugs and other substances added to the perfused medium.
It has been known for over 150 years that bone responds to mechanical loading. Although the effects of exercise and mechanical loading on the musculoskeletal systems have been well documented, the actual mechanisms by which mechanical loading acts at the cellular level in the maintenance of skeletal integrity are not completely understood. Although greater attention is being given to exercise and nutrition as a means of preventing and/or treating osteoporosis, the regulatory mechanisms that control skeletal response to mechanical loading, growth factors and nutrition are not yet delineated.
There is speculation about the biophysical structure and properties of the sensory and biochemical and molecular biological mechanism of mechano-transduction. When controlled loads of a given magnitude and frequency are applied, in vivo, either in an isolated wing preparation or a rat tibia, bone mineral density is known to increase to an extent which is approximately proportional to the load applied. However, according to the prior art, it is not possible to assess quantitatively the bone-specific regulatory control product and their mechanisms nor to monitor the bone production of local growth factors and cytokines, in these in vivo preparations.
Whilst cell culture preparations do permit an investigator to quantify second messengers, cytokines and local growth factors, they do not permit one to monitor the responses of bone cells to mechanical deformation of the bone matrix which are so important in maintaining and/or remodeling of the skeletal system.
Although growth factors have been shown to enhance the development of new bone, clearly and without the presence of mechanical loading, under these circumstances, the new matrix is not formed along lines of strain and it is that feature, in life, which induces maximum integrity of the new bone so formed. The present authors have been associated with previous work in which the viability of osteoblasts from 2 to 4 week old pigs was successfully maintained, in culture, for 68 days. Careful consideration of these findings led to the hypothesis that, in a suitable novel system, which would permit continuous perfusion and mechanical loading of suitable explanted samples of trabecular bone from mature pigs, viability might be maintained for 10 to 12 days or longer. If this were to be achieved, such a time frame would permit measurements of the rate of bone formation and resorption of the trabecular bone, not available using the systems, apparatus and methods of the prior art. Further, such a novel system would be applicable to the study of human bone.
Up to now, prior art apparatus and systems for investigating bone have either comprised cell culture apparatus of a variety of well-known types or mechanical means for applying three point and four point bending forces to a biological test subject. An example of the three point type is disclosed in U.S. Pat. No. 5,406,853 to Lintilhac and Vesecky and an example of the four point type is disclosed in U.S. Pat. No. 5,383,474 to Recker and Akhter.
The present authors are not aware of any prior art system or apparatus which provides means for simultaneous, contemporaneous and continuous study of axially loaded viable mammalian bone undergoing concurrent continuous perfusion and the effluent medium therefrom.
References
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In the instant system, apparatus means is provided for the perfusion and axial mechanical loading of an explanted sample of mammalian trabecular bone which has been prepared in an appropriate manner. During use, a prepared trabecular bone biopsy core is placed within the apparatus and is then loaded mechanically to induce tension and/or compression to the bone matrix. The bone explant perfusion and loading apparatus of the instant system is provided with means for maintaining an environment with stable oxygen, carbon dioxide, nutrients and systemic hormones.
Prepared bone biopsies are in the form of trabecular bone cores, 10-12 mm in diameter and 3 to 5 mm thick. These are surgically extracted, under sterile conditions, from suitable long bones of the subject. This procedure is carried out with care and precision, using suitable cutting means and cooling means, to ensure that the resultant bone disk samples are not subjected to temperature rises during cutting and that extreme dimensional accuracy and disk flatness are achieved.
Cutting means are in the form of a surgical hand saw used to cut gross samples, a diamond tipped keyhole saw to remove bone cores from the gross samples and an ultra high precision band saw with a diamond tipped bladed, operated in conjunction with jig means, to cut bone disk. Trabecular bone sample disks produced are flat (xc2x1100 nm) and have parallel end surfaces (xc2x12-5 cm). Cooling means comprise suitable phosphate buffered saline (PBS) at 6xc2x0 C. which is used to flood the work piece during cutting. Each trabecular bone sample disk, so prepared, is intended to supply about 3,500,000-11,000,000 cells (based on an estimate of 10,000-20,000 cells per cubic mm of bone (Mundy 1990; Parfitt 1983). Extracted bone disk samples are perfused and maintained with suitable circulating medium, Hepes and fetal calf serum.
The apparatus of the instant invention provides means for concurrent mechanical loading of the prepared bone explant sample disk, located within a novel perfusion chamber, in a controlled manner. The maximum compressive strain applied to each sample is 0.5% (5,000 xcexcE) generally at 1 Hz, with the capability of using steeper rise times, if desired. These figures translate to a maximum compression, in each sample, of 20 xcexcm, at a rate of 50,000 xcexcE secxe2x88x92. Further, the apparatus applies to the bone sample disks, controlled deformations of 200 nm. The apparatus applies forces of up to 800N, at frequencies in the physiological range, of up to 15 Hz and maximum strain rates of between 10,000 xcexcE secxe2x88x921 and 50,000 xcexcE secxe2x88x921. These data are appropriate to samples of spongy mammalian bone in which Young""s modulus varies between 400 MPa and 1200 Mpa.
The apparatus of the instant system also provides an environment in which many factors can be investigated. Because whole tissue is used, bone cells can be studied in a near-natural environment of bone matrix and bone marrow. The apparatus provides means for the user to monitor cellular response but additionally and in a novel manner, to monitor the architecture, strain characteristics and strength of the bone disk and changes therein.
The bone explant perfusion and mechanical loading apparatus of the present invention preserves the hard matrix of the bone sample and permits the collection of second messengers and growth factors in the perfusion medium. The instant system thus has many of the advantages of cell culture, whilst retaining the bone matrix encountered in vivo.
Means provided within the instant system permit recording of changes in the explanted trabecular bone core sample and further permit the calculation of strain, load and Young""s modulus for each such sample. Thus, the instant system permits not only the monitoring of second messengers, cytokines and growth factors but further permits study of how these factors, in conjunction with mechanical loading, will maximize skeletal response to varied stimuli both alone and in combination.
In the instant system there are provided perfusion loading apparatus means, power means, control means, computer hardware means, software means and sampling and analysis methods.
The perfusion loading apparatus comprises frame means, adjustable biasing pre-loading means, translator loading means, force sensor means and perfusion chamber means. Most components are substantially cylindrical and are accurately machined in corrosion resistant metal, conveniently stainless steel.
Frame mounting means are in the form of a relatively massive cylindrical frame housing, comprising a base, a lower frame section, an upper frame section and a cap, each adapted to fit together. These components are secured together with a series, conveniently of 6, partially male-threaded hardened steel bolts which pass through the frame components and are each tightened down with a female-threaded nut. The frame is about 150 mm high and about 80 mm in diameter. The lower part of the frame is substantially solid and has an axial cylindrical hole to accept a ceramic stacked piezo translator which is secured in place by virtue of a close fit in the lower frame and also by screw means through the base.
The top part of the frame provides mounting means for adjustable biasing pre-loading means provided by adjustable screw means located axially in and through and the frame cap and secured thereto by threaded means. Within the adjustable biasing pre-loading means there is provided locating and bearing means for force sensor means in the form of an annular quartz crystal force sensor in a precision welded housing.
The perfusion chamber assembly is located axially and centrally in the upper section of the frame and comprises a stainless steel bottom bearing cap which provides mounting means for a perfusion chamber body made in durable biologically inert, non-leaching plastics, preferably polycarbonate. A piston, conveniently made in stainless steel, is provided with sealing means in the form of an xe2x80x98Oxe2x80x99 ring, made from resilient and biologically inert material, preferably neoprene, engages with the upper part of the perfusion chamber body and under the influence of the pre-loading and loading entities, bears down upon a cylindrical explanted trabecular bone sample placed therein. Fluid pathways formed in the perfusion chamber body are disposed so as to ensure that perfusing fluid reaches all parts of the bone sample. Spigots provide connecting means for suitable tube means for delivering perfusing fluid to the assembled perfusion chamber and for collecting effluent from it.
The upper and lower components of the perfusion chamber are provided with locating and compression centering means and the assembly is located axially above and upon the translator loading means and directly beneath and in contact with the adjustable pre-load means which drive through push rod and ball bearing coupling means.
The piezo translator is provided, via cable connecting means, with a suitable control interface having a microprocessor controlled digital to analogue converter, low voltage driver, controller and power supply, a high voltage amplifier and display unit, all having performance and operating characteristics appropriate to the functional applications of the instant system.
The force sensor is provided, via cable connecting means, with a suitable force amplifier having an appropriate power supply and display unit, all having performance and operating characteristics appropriate to the functional applications of the instant system.
It will now be apparent that frame means, in co-operation with adjustable biasing pre-load means having force sensor means, translator loading means and perfusion chamber means, as hereinbefore described, constitute perfusion means and instrumented axial press means for the perfusion and mechanical loading of an explanted human or animal bone sample.
An explanted trabecular bone sample, prepared as hereinbefore described, is placed within the perfusion chamber, which is then assembled to the frame and loading apparatus. With connections established, power on, and perfusing fluid flowing, the adjustable biasing pre-loading sub-system is adjusted to remove lost motion from and to apply a biasing force to the load train. The biasing force is applied using a large load adjustment knob situated above the frame which drives the adjustable biasing pre-loading means via fine-threaded screw means. A suitable biasing pre-load may also be established using electro-mechanical means via regulator loop means provided in the translator controller. Establishment of a biasing force allows system integrity to be checked. The desired working load or linear translation for the experiment in hand may then be effected using the translator and translator controller.
Serial samples of effluent may be collected and assayed for one or more selected factors. Voltage outputs from the translator and charge output from the force sensor are processed and displayed visually. These are used for input to a suitable standard personal computer employing a standard operating system and running a bespoke software program for manipulating data. The program provides software means which produce outputs, via a standard interface, to the system for set-up, configuration, calibration and control of hardware as well as for calculation of relaxation and Young""s modulus. Numerical and graphical results may be output to a suitable monitor and printing device connected to the computer.
The instant system allows assessment of bone cellular response to specific stimuli, under controlled conditions. An understanding of these mechanisms will allow their manipulation which may possibly lead to the alleviation or control of osteoporosis and other deleterious skeletal changes. The instant system advances the state art in permitting investigators to study physiological responses of bone tissue under specified conditions. The instant system also advances the state of the art in permitting study of human bone biopsies in a controlled environment. It provides means for identifying morphologic changes occurring in different bone diseases and potentially, for the determination of the physiologic and genetic determinants in such diseases.
It is thus a first and most important object of the present invention to provide a novel system for continuous perfusion in conjunction with mechanical loading and for collecting and monitoring second messengers, cytokines and growth factors produced by a viable explanted bone sample in order to study skeletal response to varied stimuli both alone and in combination.
It is a second important object of the present invention to provide novel means within the instant system for recording changes in thickness of an explanted bone sample during mechanical loading and further for the calculation of strain, load and Young""s modulus for each such sample.
It is a third important object of the present invention to provide novel apparatus means for concurrent perfusion and axial mechanical loading of an explanted sample of mammalian bone, prepared in an appropriate manner, for an extended period during which the bone is to be kept viable.
It is a fourth object of the present invention to provide suitable control and recording means for novel apparatus means for concurrent perfusion and axial mechanical loading of an explanted sample of mammalian bone.
The instant system will now be described in more detail in conjunction with the following drawings.